Dry Mouth, Focal Lymphocytic Sialadenitis, and Oral Dysbiosisin IkB-z-Deficient Mice

Sjögren;;s syndrome (SS);focal lymphocytic sialadenitis (FLS);salivary flow rate;oral microbiota;IkB-z-deficient mice;617.6

면역 및 분자미생치의학 전공 ;

University:서울대학교 대학원

关键词: Sjögren;    ;    s syndrome (SS);    focal lymphocytic sialadenitis (FLS);    salivary flow rate;    oral microbiota;    IkB-z-deficient mice;    617.6;   

Others  :  http://s-space.snu.ac.kr/bitstream/10371/161684/1/000000158425.pdf

美国|英语

来源: Seoul National University Open Repository

【 摘 要 】

BackgroundSjögren syndrome (SS) is a systemic autoimmune disease characterized by dryness of eyes and mouth. Etiopathogenetic mechanisms of SS remain elusive because numerous factors contribute to the progression of SS. It is believed that the cause of SS involves a combination of genetic and environmental factors. Mice lacking IkB-z, a protein encoded by Nfkbiz gene, would be an appropriate animal model for SS because genetic and environmental factors are involved in the model. IkB-z-deficient epithelial cells cause changes in the transcriptional activity of NF-kB, leading to increased caspase-3 processing and apoptosis. Damaged epithelia fail to protect from the external environment. It is more accessible to encounter the pathogens and their substances. Moreover, a previous study demonstrated that IkB-z-deficient mice spontaneously develop SS-like autoimmune disease, resulting in increased autoantibody production, reduced tear secretion and lymphocyte-infiltrated dacryoadenitis. Although the previous study reported no symptom in the salivary glands of IkB-z-deficient mice, reduced salivary rates and sialadenitis were observed in my animal facility.MethodsIkB-z-deficient mice were bred and maintained under specific-pathogen-free condition in Laboratory Animal Facility at the School of Dentistry, Seoul National University. Five of each Nfkbiz+/+, Nfkbiz+/- and Nfkbiz-/- mice were used in the experiment. Salivary glands and oral microbiota were obtained at 24 weeks. Salivary flow rate was measured for 10 min after pilocarpine injection. Salivary glands were harvested, fixed with 10% neural formalin solution, and embedded with paraffin. They were subjected to staining with hematoxlin-eosin (H&E) or in-situ hybridization using a universal probe for bacterial 16s ribosomal RNA (rRNA). Focal lymphocytic sialadenitis (FLS) and bacterial invasion were examined under a microscope. The other pieces of salivary glands were embedded in OCT compound and then frozen in liquid nitrogen. The sections were stained with anti-mouse CD3-z and anti-mouse B220 antibodies. Immunofluorescence images were captured under a confocal microscope. Oral microbiota was collected with clean cotton swabs. Genomic DNA isolated from oral swab was subjected to microbiota analysis by high throughput sequencing.ResultsBoth Nfkbiz+/- and Nfkbiz-/- mice produced less saliva than Nfkbiz+/+ mice in response to pilocarpine injection. FLS area and foci score tended to increase in Nfkbiz-/- mice compared to other genotypes, but statistical significance was not achieved. Importantly, three out of five IkB-z-deficient mice developed FLS with score ≥ 1. Immunohistofluorescent detection with anti-mouse CD3-z and anti-mouse B220 antibodies demonstrated that infiltrating lymphocytes were mostly T cells in the early stage. Afterwards, there was a large mass of B cells occupying salivary glands. In addition, in-situ hybridization using a universal probe for 16S rRNA revealed that bacterial invasion of ductal cells occurs in submandibular glands. Oral microbiota was analyzed in Nfkbiz+/+, Nfkbiz+/- and Nfkbiz-/- mice. In alpha diversity, Simpson (measure of evenness) and Shannon (measure of species richness and evenness) indexes showed significant differences between Nfkbiz+/+ mice and Nfkbiz-/- mice. Beta diversity revealed that oral bacterial communities in Nfkbiz+/+ mice and Nfkbiz+/- mice clustered closely; on the other hand, oral bacterial communities in Nfkbiz-/- mice were separated from those of other genotypes. While Streptococcus danieliae predominated the oral microbiota in Nfkbiz+/+ and Nfkbiz+/- mice, it was substantially decreased in Nfkbiz-/- mice. Instead, Staphylococcus sciuri group, Escherichia coli group, Enterococcus faecium group and Corynebacterium mastitidis group were significantly increased in Nfkbiz-/- mice compared to Nfkbiz+/+ mice. Surprisingly, those bacteria higher in Nfkbiz-/- mice significantly increased in FLS score of ≥ 1. Nfkbiz+/- mice had unique sets of oral bacteria, which were Lactobacillus mudanjiangensis, Lactobacillus parabrevis group, Lactobacillus brevis, Lactobacillus plantarum group, Staphylococcus aureus group, Cupriavidu metallidurans and Lactobacillus_uc. Those oral bacteria were also significantly higher in the groups that were below the salivary flow rate of 7.8 (mean).ConclusionIn conclusion, Nfkbiz-/- mice spontaneously developed dry mouth, FLS, and dysbiosis of oral microbiota. Oral microbiota analysis demonstrated that the groups Staphylococcus sciuri, Escherichia coli, Enterococcus faecium, and Corynebacterium mastitidis may be associated with FLS. These findings may provide valuable information pertaining to the combined effects of host genotype and oral microbiota acting in conjunction with environmental factors to develop dry mouth and FLS.